Vacuole-inducing compounds, methods of making and using the same

ABSTRACT

Vacuole- or exosome-inducing indole-based chalcone and substituted triazole-hydrazone compounds that induce endosomal vacuolization and increase exosome yield, but do not trigger growth arrest or cell death, and methods of making and using are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 62/458,660 filedFeb. 14, 2017, the entire disclosure of which is expressly incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with U.S. Government support under grant numberR01 CA114595 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Exosomes are vesicles with diameters in the range of 30-120 nm, whichare released from many types of cells. Exosomes originate asintralumenal vesicles (ILVs) within multivesicular late endosomes(MVEs). The vesicles are released into the extracellular environmentupon fusion of MVEs with the plasma membrane. MVEs are commonly viewedas occupying an intermediate position between early endosomes andlysosomes in the endolysosomal degradative pathway of eukaryotic cells.Several distinct multiprotein ESCRTs (endosomal sorting complexesrequired for transport) function in MVE biogenesis. Proteins displayingmonoubiquitin signals interact with the ESCRTs and are sorted into theintralumenal vesicles, which are ultimately degraded when the MVEs mergewith lysosomes. While exosomes contain proteins typically found in ILVs,there is some evidence that they may be derived from functionallydistinct subpopulations of MVEs that are routed to the plasma membraneinstead of the lysosomes. The mechanisms that control the trafficking ofMVEs to the cell surface and the release of exosomes are not wellunderstood. Nevertheless, several common factors appear to influencethis process, including ceramide levels, intracellular calcium,macroenvironmental pH and specific Rab GTPases.

Despite intense interest in the potential diagnostic and therapeuticapplications of exosomes, small molecules that can either inhibit orstimulate exosome production without affecting cell growth or viabilityare lacking. Many compounds that perturb endolysosomal vesicletrafficking and induce vacuolization of late endosomal compartments havebeen identified. However, little is known about the possible effects ofsuch compounds on exosome biogenesis because many of them are cytotoxic.

It would be useful to have small molecules that can either inhibit orstimulate exosome production without affecting cell growth or viability.

SUMMARY OF THE INVENTION

Provided herein a method for increasing exosome production in a cell byexposing the cell to an indole-pyridinyl-propenone compound that inducesendosomal vacuolization but does not trigger growth arrest or celldeath.

In certain embodiments, the indole-pyridinyl-propenone compound has thefollowing chemical structure:

where R═OH, OCH₃, OCH₂CH₃, OCH(CH₃)₂, NHCOCH₃ or NHCO₂C(CH₃)₃; and

R′═H, CH₃, CH₂CH₂CH₃, CH(CH₃)₂, or CH₂CH(CH₃)₂,

excluding where R═OCH₃ and R′═CH₃.

In certain embodiments, the indole-pyridinyl-propenone compound has theformula where: R═OCH₃; and, R′═H, CH₂CH₂CH₃, CH(CH₃)₂, or CH₂CH(CH₃)₂.

In certain embodiments, the indole-pyridinyl-propenone compound has theformula where: R═OCH₃, and R′═CH₂CH₂CH₃ (i.e.,3-(5-methoxy-2-propyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(MOPIPP).

In certain embodiments, the indole-pyridinyl-propenone compound has theformula where: R═OH, OCH₂CH₃, OCH(CH₃)₂, NHCOCH₃ or NHCO₂C(CH₃)₃; and,R′═CH₃.

In certain embodiments, the indole-pyridinyl-propenone compound has theformula where: R═OCH₂CH₃; and, R′═CH₃.

In certain embodiments, the indole-pyridinyl-propenone compound has theformula where: R═OCH₂CH₃; and R′═CH₂CH₂CH₃.

In another aspect, there is provided a method for increasing exosomeproduction in a cell culture, comprising: exposing the culture to asubstituted triazole-hydrazone compound that induces endosomalvacuolization but does not trigger cell death or attenuate cell growth.

In certain embodiments, such substituted triazole-hydrazone compound hasthe following chemical structure:

wherein R═CH₂, NH, O; and R′=an electron withdrawing group. Non-limitingexamples of electron withdrawing groups include: halides (F, Cl, Br andI), trifluoromethyl (CF₃), nitro (NO₂), acetyl (—COCH₃) andtrimethylammonium (N(CH₃)₃ wherein this becomes positively charged whenfurther attached as a substituent. Also, in certain embodiments, thesubstituted triazole-hydrazone compound has the formula where R═O; andR′=Iodo.

Also described herein is a method where exosomes are collected exosomesby one or more of the following: ultracentrifugation; columnchromatography; size exclusion; and filtration through a devicecontaining an affinity matrix that is selective toward exosomes.

Also described is a method using such exosomes by harvesting theexosomes for use as nanocarriers for packaging and delivery of atherapeutic material to a subject in need thereof.

In certain embodiments, the therapeutic material comprises one or moreof: small molecules, mRNAs, and proteins.

In certain embodiments, the cell comprises one or more of: multipotentmesenchymal stromal cells; stem cells; and, cancer cells. Further, incertain embodiments, the cell is a mammalian cell.

Also described herein is a method of producing transiently expressedmiRNA containing cells, comprising: transfecting a cell population ofcells capable of producing exosomes produced according to the methoddescribed herein with one or more plasmids encoding miRNA; harvestingcells from the cell population after transfection; and, confirming thepresence of the miRNA in one or more of the harvested cells.

Also described herein is a method of treating a subject in need thereofwith modified exosomes, comprising: transfecting exosome-producing cellsproduced by the method described herein, with one or more carriersencoding a therapeutic material; harvesting the exosomes from the cellpopulation or media containing same after transfection; confirming thepresence of the therapeutic material in one or more of the harvestedexosomes; and, administering to the subject one or more of the harvestedexosomes in a pharmaceutically effective amount to treat the subject.

Other systems, methods, features, and advantages of the presentinvention will be or will become apparent to one with skill in the artupon examination of the following drawings and detailed description. Itis intended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIGS. 1A-1D—Comparison of cell and exosome morphologies in culturestreated with MOPIPP and vacuolin-1. (FIG. 1A) U251 cells were examinedby phase contrast microscopy after 24 h treatment with 10 μM MOPIPP, 1μM vacuolin-1 or an equivalent volume of DMSO (control). Scale bars=20μm. (FIG. 1B) Electron micrographs of U251 cells after 24 h treatmentwith MOPIPP show many large vacuoles (v), with some containing clustersof ILVs (asterisks). Scale bars=500 nm. (FIG. 1C and FIG. 1D) Exosomesisolated from cells treated for 24 h with the indicated compounds werecharacterized by electron microscopy (FIG. 1C) or DLS (FIG. 1D).

FIGS. 2A-2D—MOPIPP and vacuolin-1 increase the amounts of exosomalmarker proteins in vesicle fractions recovered from conditioned medium.In three separate experiments, U251 cells were treated for 24 h with 10μM MOPIPP, 1 μM vacuolin-1 or an equivalent volume of DMSO vehicle. Thecells from each experiment were counted (mean±SEM) (FIG. 2A), and themedium from the same cultures was used to prepare exosomes with theExo-spin™ Purification method. Equal aliquots of the final exosomepreparations were subjected to western blot analysis for the indicatedproteins (FIG. 2B and FIG. 2C). In FIG. 2C, representative blots areshown, and the fold-increase in the treated cells relative to thecontrols is graphed below each blot (mean±SEM). The cells from the sameexperiments (see FIG. 2A), were subjected to western blot analysis forthe indicated proteins, with equal amounts of protein loaded on eachlane (FIG. 2B and FIG. 2D). In FIG. 2D, representative blots are shown,with signals for the proteins in the treated cells expressed as percentof the corresponding DMSO controls in the graphs below each blot(mean±SEM).

FIGS. 3A-3D—MOPIPP or vacuolin-1 do not have major effects on theprofile of miRNAs represented in exosomes. (FIG. 3A) Cellular miRNAsexpressed in untreated U251 cells were profiled using the Human BrainCancer miScript® arrays (n=3) and ΔCt values were determined using thesix snoRNA/snRNA miScript PCR controls included on each array. Thespecific miRNAs selected for further study are indicted with arrows.(FIG. 3B) U251 cells were treated with 10 μM MOPIPP or an equivalentvolume of DMSO for 24 h and cellular expression of each of the indicatedmiRNAs was determined by RT-PCR (n=3). (FIG. 3C) In three separateexperiments, exosomes were isolated from U251 cells treated with MOPIPP,vacuolin-1 or DMSO and equal amounts of exosomal RNA were subjected toreverse transcription and RT-PCR to assess miR-21-5p. (FIG. 3D) RT-PCRcomparisons of five additional miRNAs, starting with equal amounts ofexosomal RNA from cells treated with MOPIPP, vacuolin-1 or DMSO.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Definitions

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to texts, journal referencesand contexts known to those skilled in the art.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers and enantiomers of the group members, are disclosed separately.When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.It is intended that any one or more members of any Markush group orlisting provided in the specification can be excluded from the inventionif desired. When a compound is described herein such that a particularisomer or enantiomer of the compound is not specified, for example, in aformula or in a chemical name, that description is intended to includeeach isomers and enantiomer of the compound described individual or inany combination. Additionally, unless otherwise specified, all isotopicvariants of compounds disclosed herein are intended to be encompassed bythe disclosure. For example, it will be understood that any one or morehydrogens in a molecule disclosed can be replaced with deuterium ortritium. Isotopic variants of a molecule are generally useful asstandards in assays for the molecule and in chemical and biologicalresearch related to the molecule or its use. Specific names of compoundsare intended to be exemplary, as it is known that one of ordinary skillin the art can name the same compounds differently.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing components that aredescribed in the publications that might be used in connection with thepresently described invention.

The definitions are provided to clarify their specific use in thecontext of the invention.

Additionally, described herein are pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents. The phrase, “pharmaceutically acceptable derivative”, as usedherein, denotes any pharmaceutically acceptable salt, ester, or salt ofsuch ester, of such compound, or any other adduct or derivative which,upon administration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof.

The term “stable”, as used herein, preferably refers to compounds whichpossess stability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

As used herein, the terms “patient” and “subject” are intended toinclude living organisms in which certain conditions as described hereincan occur. Examples include humans, monkeys, cows, sheep, goats, dogs,cats, mice, rats, and transgenic species thereof. In a preferredembodiment, the patient is a primate. In an even more preferredembodiment, the primate is a human. Other examples of subjects includeexperimental animals such as mice, rats, dogs, guinea pigs, cats, goats,sheep, pigs, and cows. The experimental animal can be an animal modelfor a disorder, e.g., a transgenic mouse with cancer.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

General Description

Various indolyl pyridinyl propenones are described in Maltese et al.U.S. Pat. No. 9,023,871 issued May 5, 2015; Maltese et al. U.S. Pat. No.9,028,796 issued May 12, 2015; Maltese et al. U.S. Pat. No. 9,061,994issued Jun. 23, 2015; Trabbic et al., “Differential Induction ofCytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-SubstitutedPyridinylpropenones,” ACS Med. Chem. Lett., 5 (2014) 73-77; and, Trabbicet al., “Synthesis and biological evaluation ofindolyl-pyridinyl-propenones having either methuosis or microtubuledisruption activity,” J. Med. Chem., 58 (2015) 2489-2512, the entirecontents of which are explicitly incorporated herein.

Various indolyl pyridinyl propenone compounds induce the formation ofcytoplasmic vacuoles in cultured human cells without causing appreciablecytotoxicity or growth arrest.

Described herein are methods using the vacuole-inducing analogs of theindolyl pyridinyl propenone chemical family to stimulate an increase inexosome production in cultured cells without altering the cargo carriedby these exosomes.

Also described herein are methods using the vacuole-inducing analogs ofthe indolyl pyridinyl propenone chemical family to stimulate an increasein exosome release by cultured cells without altering the cargo carriedby these exosomes.

Also described herein are therapeutic applications using such exosomes;for example, using these exosomes as nanocarriers for packaging anddelivery of a desired cargo to the cell. In certain embodiments, thecargo can include small molecules (e.g., anti-cancer drugs), miRNAs, andproteins.

These exosomes are useful in that they may incorporate various cargoesin producer cells.

The methods of production of these exosomes overcomes one of the majorimpediments to more widespread development of exosome-based therapies;that is, the low yield of exosomes.

For example, one compound3-(5-methoxy-2-propyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(MOPIPP), and related molecules are useful as non-cytotoxicpharmacological agents to boost exosome production.

MOPIPP and related molecules are useful in enhancing large-scalecommercial production of exosomes from cultured cells, particularly fortherapeutic purposes.

The following examples show the use of cultured human glioblastoma cellsto assess the effects of MOPIPP and vacuolin-1 on exosome production.The results show that both compounds may have utility as non-toxicagents to enhance the cellular release of exosomes.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. All publications, including patents and non-patentliterature, referred to in this specification are expressly incorporatedby reference. The following examples are intended to illustrate certainpreferred embodiments of the invention and should not be interpreted tolimit the scope of the invention as defined in the claims, unless sospecified.

It is to be understood, that throughout the specification and claimsherein, the terms “vacuole,” “vesicle” and “exosome” generally refer toa space within the cytoplasm of a cell, enclosed by a membrane andtypically containing fluid, which may also be released from the cell.For example, exosomes are generally described as small vesicles createdand released from the plasma membrane of various types of cells,especially immune cells, and capable of inducing antigen-specific immuneresponses.

Materials and Methods

Cell Culture

U251 human glioblastoma cells were purchased from the DCT TumorRepository (National Cancer Institute, Frederick, Md.). Stock cultureswere maintained in Dulbecco's modified Eagle medium (DMEM) containing10% (v/v) fetal bovine serum (FBS) (JR Scientific, Woodland, Calif.) at37° C. with an atmosphere of 5% CO₂ in air. Cultures were passaged forless than 6 months and were monitored periodically for Mycoplasmacontamination.

Isolation of Exosomes

Prior to beginning studies, cells were seeded in 10 cm diameter culturedishes at 500,000 cells/dish and maintained for 24 h in DMEMsupplemented with 10% Gibco™ exosome-depleted FBS (ThermoFisherScientific, Waltham, Mass.). The medium was then replaced with freshmedium containing either 10 μM MOPIPP (synthesized as described inTrabbic et al., “Differential Induction of Cytoplasmic Vacuolization andMethuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones, ACS Med.Chem. Lett., 5 (2014) 73-77) or 1 μM vacuolin-1 (Santa CruzBiotechnology, Santa Cruz, Calif.) dissolved in DMSO. Control culturescontain an equivalent volume of the DMSO vehicle (0.1%) in the medium.After 24 h, the medium was collected from the dishes (typically 10-12dishes per condition) and the attached cells were pooled and countedwith a Coulter Counter (model Z1). Exosomes were isolated from mediumusing the Exo-spin™ Exosome Purification system (Cell Guidance Systems,St. Louis, Mo.). The medium was pre-cleared by centrifugation at 4° C.,first at 300×g for 10 min and then at 16,000×g for 30 min. Then a volumeof Buffer A equal to half the volume of medium was added and the mixturewas incubated overnight at 4° C. The precipitate, enriched withexosomes, was collected by centrifugation at 16,000×g for 1 h, and thepellet was re-suspended in 100 μl of Dulbecco's phosphate-bufferedsaline (PBS), pH 7.4. The material was applied to an Exo-Spin columnequilibrated with PBS, and the purified exosomes were eluted in 200 μlof PBS.

Electron Microscopy

Aliquots of purified exosomes obtained from control and drug-treatedcell cultures were fixed with 4% paraformaldehyde and stained with 2%uranyl acetate on Formvar carbon-coated electron microscopy grids.Vesicles were visualized using a Hitachi HD-2300 transmission electronmicroscope at an accelerating voltage of 200 kV. Electron microscopy ofvacuolated cells was carried out (as described in Johnson et al. “Genesilencing reveals a specific function of hVps34 phosphatidylinositol3-kinase in late versus early endosomes, J. Cell Sci., 119 (2006)1219-1232).

Dynamic Light Scattering (DLS)

Exosomes suspended in PBS were subjected to DLS using a Nicomp 380 ZLSinstrument (Particle Sizing Systems, Port Richey, Fla.). Samples wereplaced in the path of a helium neon laser of wavelength 658 nm at 23°C., and data were collected at a scattering angle of 90°. For eachsample, three measurements of 8 min each were performed, and theparticle size distribution (number-weighted diameter) was displayed.

Immunoblot Analysis

The cells in the dishes used for collection of exosomes were washedthree times with PBS and lysed in SDS sample buffer (Laemmli, “Cleavageof structural proteins during the assembly of the head of bacteriophageT4, Nature, 227 (1970) 680-685). The protein concentration wasdetermined by colorimetric assay using Bio-Rad reagent (Bio-Rad,Richmond, Calif.). Samples containing equal amounts of total cellprotein were subjected to SDS-PAGE and immunoblot analysis (as describedin Maltese et al., “Retention of the Alzheimer's amyloid precursorfragment C99 in the endoplasmic reticulum prevents formation of amyloidbeta-peptide, J. Biol. Chem., 276 (2001) 20267-20279). For analysis ofproteins in exosomes, equal volumes of purified exosomes isolated fromcontrol or treated cells were mixed with ⅕ volume of 5×SDS sample bufferprior to SDS-PAGE. Monoclonal antibodies against CD63 and LAMP-1 wereobtained from the Developmental Studies Hybridoma Bank (Iowa City,Iowa). Antibodies against Alix and cytochrome c were from Santa CruzBiotechnology (Santa Cruz, Calif.), and the antibody against Lamin B₂was from Cell Signaling Technology (Danvers, Mass.). HRP-coupled goatanti-mouse IgG was from BD Biosciences (San Jose, Calif.).Chemiluminescent signals on the membranes were quantified using an AlphaInnotech FluorChem HD2 imaging system with Alpha View software.

Analysis of miRNAs

Total RNA was extracted and purified from cultured cells or exosomesusing QIAzol™ lysis reagent followed by RNAeasy Mini spin-columns,according to the manufacturer's protocol (SA Biosciences/Qiagen,Germantown, Md.). For each sample, cDNA was generated by reversetranscription of 150 ng of total RNA, using the miScript II RT kit(Qiagen). RNA and cDNA were quantified and checked for purity (OD260/280) using a Nano-Drop -1000 spectrophotometer (ThermoFisher). Forinitial profiling of the miRNAs expressed in control or MOPIPP-treatedcells, equal amounts of cDNA were applied to Human Brain CancermiScript® miRNA PCR arrays (MIHS-108Z) (SA Biosciences/Qiagen), andreal-time PCR reactions were carried in an Applied Biosystems StepOnePlus™ system using SYBR Green master mix. Raw Ct values for theindividual miRNAs were normalized to the average Ct value for sixsnoRNA/snRNA miScript PCR controls included on each array, yielding ΔCtvalues. For comparisons of the miRNA contents of exosomes collected fromthe medium of cells treated with MOPIPP, vacuolin-1 or vehicle (DMSO),individual miScript primers were purchased for six of the most highlyexpressed miRNAs detected in U251 cells (SA Biosciences/Qiagen). Theprimers were reconstituted in SYBR green master mix and combined withcDNAs derived from equal amounts of exosomal RNA (150 ng). RT-PCRreactions were carried out in triplicate to obtain Ct values for eachmiRNA.

Results

Characterization of Exosomes

Treatment of cultured U251 glioblastoma cells with MOPIPP resulted invacuolization of endocytic compartments. The accumulation of numerousvacuoles was readily detected by phase contrast microscopy (FIG. 1A).Treatment with vacuolin-1 induced a very similar phenotype (FIG. 1A).Despite the extreme vacuolization, the cells treated with both compoundsremained attached to the culture dishes and continued to proliferate.Electron microcopy revealed that most of the vacuoles in the cellstreated with MOPIPP were surrounded by a single membrane and werelargely devoid of intralumenal contents (FIG. 1B). However, a distinctsubpopulation of vacuoles (approximately 20-30%) contained clusters ofheterogeneous vesicles, many of which were of a size (<50 nm) consistentwith ILVs and exosomes (examples shown in FIG. 1B).

When 24-h conditioned media were collected from control and drug-treatedcultures and subjected to a procedure designed to yield purifiedexosomes, electron microscopy showed that the final exosome fractionconsisted mainly of vesicles with diameters of approximately 25-30 nm(FIG. 1C), matching the lower end of the size range reported forexosomes. There were no discernable differences in the morphologies ofthe vesicles obtained from the treated cells compared to the control.Particle analysis by DLS confirmed that the purified vesicle populationshad unimodal distributions with mean diameters in the range of 18-27 nm.Differences between control and treated cells were not statisticallysignificant (FIG. 1D).

Quantification of Exosome Marker Proteins

To determine if treatment with MOPIPP and/or vacuolin-1 might alter theproduction of exosomes, conditioned medium was pooled from 10-12 controlor treated cultures and exosomes were isolated as described above. Thecells from the same cultures were also harvested and pooled. FIG. 2Ashows that comparable numbers of cells were present in the control anddrug-treated cultures, consistent with the lack of growth inhibition byMOPIPP and vacuolin-1. The immunoblots in FIG. 2B demonstrate thatcytochrome c and lamin B₂, markers for mitochondria and nuclear enveloperespectively, were not detectable in the purified exosome populations,confirming that the latter were not contaminated with intracellularorganelles released via cell lysis. In FIG. 2C and FIG. 2D, the isolatedexosomes and cells were probed for three proteins commonly enriched inMVEs and exosomes: CD63, a member of tetraspanin protein family, Alix, aprotein involved in the biogenesis of endosomal ILVs, and LAMP-1, anabundant membrane glycoprotein in lysosomes and late endosomes.

The results show that the relative amounts of all three proteins wereincreased by several fold in the extracellular vesicle preparations fromcultures treated with MOPIPP (FIG. 2C). An even greater increase wasobserved in the cultures treated with vacuolin-1 (FIG. 2C). In contrast,changes in expression of the same marker proteins in the correspondingcell populations were comparatively modest (FIG. 2D). Since the exosomeswere isolated from nearly identical numbers of cells in the control andtreated cultures (FIG. 2A), the results show that MOPIPP and vacuolin-1promote an increase in the release of exosomes into the extracellularenvironment.

Comparison of miRNA Profiles

miRNA cargoes carried by exosomes play important roles in intercellularcommunication. As shown herein, it was then determined whether, inaddition to increasing exosome output, MOPIPP and vacuolin-1 would alterthe miRNA composition of the exosomes. miRNAs expressed in untreatedU251 glioblastoma cells were profiled, using a RT-PCR array that detects84 miRNAs commonly associated with human brain cancers. Based on theirΔCt values relative to a panel of six normalization controls (FIG. 3A),13 miRNAs with high expression were selected for further comparisonbetween control and MOPIPP-treated cells.

As shown in FIG. 3B, treatment with MOPIPP did not cause major changesin the cellular expression of any of the selected miRNAs. Among themiRNAs profiled on the array, miR-21-5p was clearly the most abundant.RT-PCR reactions were carried out with miR-21-5p primers to assess therelative amounts of miR-21-5p in exosomes collected from cells treatedwith MOPIPP or vacuolin-1, compared to untreated controls. Since theselection of a definitive RT-PCR standard for normalization of exosomalmiRNA is controversial, triplicate reactions were set up with equalamounts of total exosomal RNA from control and treated cells anddirectly compared the raw Ct values (FIG. 3C).

Using this approach for three independent experiments, there were noobservations of any changes in the relative abundance of miR-21-5-p inexosomal RNA collected from cells treated with MOPIPP or vacuolin-1.Similar results were obtained when the analysis was extended to fiveadditional miRNAs (FIG. 3D).

Discussion

The results show that vacuolization of late endosomal compartmentsinduced by two distinct small molecules, MOPIPP and vacuolin-1, isaccompanied by a several-fold increase in exosomal marker proteins inextracellular vesicles prepared from comparable numbers of glioblastomacells. Cell proliferation and viability were not markedly impaired bythese compounds, and the characteristics of the exosomes from treatedcells were similar to the controls. Since the intracellular amounts ofthe marker proteins were only modestly affected by the compounds, theresults are indicative of an increase in exosome biogenesis and/orsecretion in cells treated with MOPIPP and vacuolin-1.

Small molecules that promote vacuolization of late endosomalcompartments have the potential to affect exosome production in severalways. In one scenario, vacuolization of endosomes could disrupt themolecular machinery for the formation of ILVs, so that any MVEs thatsubsequently fuse with the plasma membrane release fewer exosomes. In asecond scenario, formation of ILVs might remain unaffected, buttrafficking of enlarged MVEs to the cell surface might be disrupted,also resulting in diminished exosome secretion. Finally, in a thirdscenario, drug-induced vacuoles derived from late endosomes could retainthe ability to generate ILVs, but be unable to merge with lysosomeswhere the vesicles are degraded. In that case, enlarged MVEs mightaccumulate and deliver more ILVs to the extracellular environment,provided that they remain competent to fuse with the plasma membrane. Itis now believed that the third scenario explains the increase in exosomesecretion (FIG. 2), considering that many vacuoles in MOPIPP-treatedcells contained numerous ILVs (FIG. 1B).

The miRNA cargo carried by exosomes may differ from the miRNA profile ofthe producer cell, implying that segregation of miRNAs into exosomes isnot random. It is now shown herein that extensive vacuolization ofendosomal compartments did not affect the expression of several commonmiRNAs in glioblastoma cells. Furthermore, the vacuole-inducingcompounds did not substantially alter the relative concentrations ofseveral miRNAs represented in the total RNA in the secreted exosomes.Thus, while the analysis of exosomal proteins shows that MOPIPP andvacuolin-1 induce large increases in exosome secretion, the exosomesappear to be qualitatively similar to those collected from untreatedcells in terms of their miRNA cargo.

Therapeutic Uses

As described herein, inventive vacuole-inducing molecules inducevacuolization in cells without cell death. In this regard, the presentvacuole-inducing molecules are useful in a number of pathologicalapplications. Certain applications are mentioned below; others will beapparent to those of ordinary skill in the art.

Therefore, vacuole-inducing molecules can be used to inducevacuolization without cell death of cancer cells. For example, thepresent are useful in treating cancers of the brain, lung, bladder,liver, spleen, pancreas, bone, colon, stomach, breast, prostate, ovary,central nervous system and skin. For example, glioblastoma and breastcarcinoma can be treated.

Further non-limiting examples of therapeutic applications of exosomesinclude using these vesicles as nanocarriers for packaging and deliveryof small molecules (e.g., anti-cancer drugs), miRNAs, and proteins.

Small molecules like MOPIPP are useful as non-cytotoxic pharmacologicalagents to boost exosome production.

Research Uses

In addition to the various pharmaceutical uses described above,vacuole-inducing compounds have utility in a variety of researchapplications, e.g., in vitro assays, including, for example, as chemicalprobes. Those of ordinary skill in the art will appreciate that thefield of chemical genetics attempts to identify chemical agents withdefinable effects on biological events, pathways, or products so thatthese agents can be used as tools to analyze the relevant biologicalevents, pathways, or products. Vacuole-inducing molecules describedherein are particularly well suited for such studies. Accordingly, suchuses also include assays, e.g., in vitro assays, utilizing thevacuole-inducing molecules to analyze vacuolization, intracellulartrafficking, antigen presentation, membrane fusion events, and relatedcellular processes. Furthermore, the vacuole-inducing molecules can alsobe used in screening assays to identify second generationvacuole-inducing molecules, e.g., molecules having modified chemicalstructures which function as vacuole-inducing molecules. Azide,propargyl or other forms of the vacuole-inducing molecules can be usedin a variety of cell-based or molecular screening assays to identifyspecific protein targets that bind to such molecules.

Formulations

As described herein, vacuole-inducing compounds can be utilized in anyof a variety of contexts, and can be formulated appropriately accordingto known principles and technologies.

For example, the vacuole-inducing molecules can be provided insubstantially pure form, in an organic solvent such as DMSO.Alternatively or additionally, vacuole-inducing molecules can beformulated as a pharmaceutical composition, for example being combinedwith a pharmaceutically acceptable carrier. It will also be appreciatedthat certain of the vacuole-inducing compounds can exist in free formfor treatment, or where appropriate, as a pharmaceutically acceptablederivative thereof. A pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a prodrug or other adduct or derivative of a compoundwhich upon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith little or no undue toxicity, irritation, allergic response and thelike, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts of amines, carboxylic acids, and othertypes of compounds, are well known in the art (see, for example, Berge,et al., 3.1. J. Pharmaceutical Sciences, 66:1-19, 1977, incorporatedherein by reference).

The salts can be prepared in situ during the final isolation andpurification of the compounds, or separately by reacting a free base orfree acid function with a suitable reagent, as described generallybelow. For example, a free base function can be reacted with a suitableacid. Furthermore, where the compounds carry an acidic moiety, suitablepharmaceutically acceptable salts thereof can, include metal salts suchas alkali metal salts, e.g., sodium or potassium salts; and alkalineearth metal salts, e.g., calcium or magnesium salts. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The pharmaceutical compositions can additionally comprise apharmaceutically acceptable carrier, which, as used herein, includes anyand all solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with the compounds, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatine; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; cornoil and soybean oil; glycols; such as propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogenfree water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

The vacuole-inducing molecules disclosed herein can be formulated oradministered together with any other known agent having a complementarybiological effect. For example, vacuole-inducing molecules can becombined with steroids or other immunomodulating agents in order toregulate immunological events as described herein.

It will be appreciated that the compounds and compositions can beadministered using any effective amount and any effective route ofadministration. Thus, the expression “effective amount” as used herein,refers to a sufficient amount of agent to result in vacuolization and/orinhibition of compartment trafficking as described herein. The exactamount required can vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of theinfection, the particular therapeutic agent, its mode of administration,and the like. The compounds are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof therapeutic agent appropriate for the patient to be treated. It willbe understood, however, that the total daily usage of the compounds andcompositions will be decided by the attending physician within the scopeof sound medical judgment.

The specific therapeutically effective dose level for any particularpatient or organism can depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, gender and diet of the patient; thetime of administration, route of administration, and rate of excretionof the specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employed;and like factors well known in the medical arts.

Dosing

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount can vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions can comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound can comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition can beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimenscan be desirable.

In other non-limiting examples, a dose can also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionscan be administered to humans and other animals orally, rectally,parenterally, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, or drops), bucally, as an oral ornasal spray, or the like, depending on the severity of the infectionbeing treated. In certain embodiments, the compounds can be administeredat dosage levels of about 0.001 mg/kg to about 50 mg/kg, from about 0.01mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 10 mg/kg ofsubject body weight per day, one or more times a day, to obtain thedesired therapeutic effect. It will also be appreciated that dosagessmaller than 0.001 mg/kg or greater than 50 mg/kg (for example 50-100mg/kg) can be administered to a subject. In certain embodiments,compounds are administered orally or parenterally.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms can contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.R and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds withsuitable nonirritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype can also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art.

In such solid dosage forms the active compound can be admixed with atleast one inert diluent such as sucrose, lactose and starch. Such dosageforms can also comprise, as in normal practice, additional substancesother than inert diluents, e.g., tableting lubricants and othertableting aids such as magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formscan also comprise buffering agents. They can optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compoundinclude ointments, pastes, creams, lotions, gels, powders, solutions,sprays, inhalants or patches. The active component is admixed understerile conditions with a pharmaceutically acceptable carrier and anyneeded preservatives or buffers as can be required. Ophthalmicformulation, ear drops, and eye drops are also contemplated as beingwithin the scope of this invention. Additionally, the present inventioncontemplates the use of transdermal patches, which have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms are made by dissolving or dispensing the compound inthe proper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate can be controlled byeither providing a rate controlling membrane or by dispersing thecompound in a polymer matrix or gel.

Kits

In other embodiments, the compounds, or compositions containing them arepackaged into a kit for conveniently and effectively carrying out themethods described herein. In general, the pharmaceutical pack or kitcomprises one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Such kits are especiallysuited for the delivery of solid oral forms such as tablets or capsules.Such a kit preferably includes a number of unit dosages, and can alsoinclude a card having the dosages oriented in the order of theirintended use. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarinsert, designating the days in the treatment schedule in which thedosages can be administered. Alternatively, placebo dosages, or calciumdietary supplements, either in a form similar to or distinct from thedosages of the pharmaceutical compositions, can be included to provide akit in which a dosage is taken every day. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated by reference herein, and for convenience areprovided in the following bibliography.

Citation of any of the documents recited herein is not intended as anadmission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

What is claimed is:
 1. A method for increasing exosome production in acell, comprising: exposing the cell to an indole-pyridinyl-propenonecompound that induces endosomal vacuolization but does not triggergrowth arrest or cell death.
 2. The method of claim 1 wherein, thecompound has the following chemical structure:

R═OH, OCH₃, OCH₂CH₃, OCH(CH₃)₂, NHCOCH₃ or NHCO₂C(CH₃)₃; and R′═H, CH₃,CH₂CH₂CH₃, CH(CH₃)₂, or CH₂CH(CH₃)₂; excluding where R═OCH₃ and R′═CH₃.3. The method of claim 2, wherein R═OCH₃; and, R′═H, CH₂CH₂CH₃,CH(CH₃)₂, or CH₂CH(CH₃)₂.
 4. The method of claim 3, wherein R═OCH₃, andR′═CH₂CH₂CH₃.
 5. The method of claim 2, wherein R═OH, OCH₂CH₃,OCH(CH₃)₂, NHCOCH₃ or NHCO₂C(CH₃)₃; and, R′═CH₃.
 6. The method of claim5, wherein R═OCH₂CH₃; and, R′═CH₃.
 7. The method of claim 2, whereinR═OCH₂CH₃; and R′═CH₂CH₂CH₃.
 8. The method of claim 1, wherein exosomesare collected by one or more of the following: ultracentrifugation;column chromatography; size exclusion; and filtration through a devicecontaining an affinity matrix that is selective toward exosomes.
 9. Amethod using exosomes produced by claim 1, comprising harvesting theexosomes for use as nanocarriers for packaging and delivery of atherapeutic material to a subject in need thereof.
 10. The method ofclaim 9, wherein the therapeutic material comprises one or more of:small molecules, miRNAs, and proteins.
 11. The method of claim 1,wherein the cell comprises one or more of: multipotent mesenchymalstromal cells; stem cells; and, cancer cells.
 12. The method of claim 1,wherein the cell is a mammalian cell.
 13. A method of producingtransiently expressed miRNA containing cells, comprising: transfecting acell population of cells capable of producing exosomes producedaccording to the method of claim 1 with one or more plasmids encodingmiRNA; harvesting cells from the cell population after transfection;and, confirming the presence of the miRNA in one or more of theharvested cells.
 14. A method of treating a subject in need thereof withmodified exosomes, comprising: transfecting exosome-producing cellsproduced by the method of claim 1, with one or more carriers encoding atherapeutic material; harvesting the exosomes from the cell populationor media containing same after transfection; confirming the presence ofthe therapeutic material in one or more of the harvested exosomes; and,administering to the subject one or more of the harvested exosomes in apharmaceutically effective amount to treat the subject.
 15. A method forincreasing exosome production in a cell culture, comprising: exposingthe culture to a substituted triazole-hydrazone compound that inducesendosomal vacuolization but does not trigger cell death or attenuatecell growth.
 16. The method of claim 15, wherein the compound has thefollowing chemical structure:

wherein R═CH₂, NH, O; and R′=an electron withdrawing group.
 17. Themethod of claim 16, wherein R═O; and R′=Iodo.
 18. The method of claim16, wherein the electron withdrawing groups are selected from: halides(F, Cl, Br and I), trifluoromethyl (CF₃), nitro (NO₂), acetyl (—COCH₃)and trimethylammonium (N(CH₃)₃.